Chen et al. Microb Cell Fact (2020) 19:32 https://doi.org/10.1186/s12934-020-01301-8 Microbial Cell Factories

RESEARCH Open Access Enhancement of production in verticillus through global metabolic regulation of N‑acetylglucosamine and assisted metabolic profling analysis Hong Chen1,2†, Jiaqi Cui1,2†, Pan Wang1,2, Xin Wang1,2 and Jianping Wen1,2*

Abstract Background: Bleomycin is a broad-spectrum glycopeptide antitumor antibiotic produced by Streptomyces verticil- lus. Clinically, the mixture of bleomycin A2 and bleomycin B2 is widely used in combination with other drugs for the treatment of various cancers. As a secondary metabolite, the biosynthesis of bleomycin is precisely controlled by the complex extra-/intracellular regulation mechanisms, it is imperative to investigate the global metabolic and regula- tory system involved in bleomycin biosynthesis for increasing bleomycin production. Results: N-acetylglucosamine (GlcNAc), the vital signaling molecule controlling the onset of development and antibiotic synthesis in Streptomyces, was found to increase the yields of signifcantly in chemically defned medium. To mine the gene information relevant to GlcNAc metabolism, the DNA sequences of dasR-dasA-dasBCD- nagB and nagKA in S. verticillus were determined by chromosome walking. From the results of Real time fuorescence quantitative PCR (RT-qPCR) and electrophoretic mobility shift assays (EMSAs), the repression of the expression of nagB and nagKA by the global regulator DasR was released under induction with GlcNAc. The relief of blmT expres- sion repression by BlmR was the main reason for increased bleomycin production. DasR, however, could not directly afect the expression of the pathway-specifc repressor BlmR in the bleomycins gene cluster. With at the beginning of bleomycin synthesis, the supply of the specifc precursor GDP-mannose played the key role in bleomycin production. Genetic engineering of the GDP-mannose synthesis pathway indicated that phosphomannose isomerase (ManA) and phosphomannomutase (ManB) were key enzymes for bleomycins synthesis. Here, the blmT, manA and manB co-expression strain OBlmT/ManAB was constructed. Based on GlcNAc regulation and assisted metabolic profling analysis, the yields of bleomycin A2 and B2 were ultimately increased to 61.79 and 36.9 mg/L, respectively. Conclusions: Under GlcNAc induction, the elevated production of bleomycins was mainly associated with the alleviation of the inhibition of BlmT, so blmT and specifc precursor synthesis pathways were genetically engineered for bleomycins production improvement. Combination with subsequent metabolomics analysis not only efectively increased the bleomycin yield, but also extended the utilization of chitin-derived substrates in microbial-based antibi- otic production.

*Correspondence: [email protected] †Hong Chen and Jiaqi Cui contributed equally to this work 1 Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, People’s Republic of China Full list of author information is available at the end of the article

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Keywords: Bleomycin, GlcNAc, DasR, GDP-mannose, Metabolic profling

Introduction defciency, which is delivered and sensed by Streptomy- Bleomycin is a member of glycopeptide antibiotics that ces to initiate sporulation and secondary metabolism was frst isolated from the fermentation broth of S. ver- [17]. Terefore, not only does GlcNAc or chitin provide ticillus by Umezawa et al. [1]. Te bleomycin gene cluster a source of carbon and nitrogen for strain growth, but and its structure clearly indicate that a hybrid peptide- the GlcNAc-induced regulation of antibiotic synthesis polyketide skeleton and a disaccharide unit together should be utilized to improve the bleomycin production constitute bleomycins with diferent C-terminal amines in S. verticillus. [2]. A mixture of bleomycin A2 and B2, marketed as After the onset of antibiotic synthesis, the supply of ­Blenoxane®, is administered by injection in clinic to treat limiting precursors is another key factor for improv- tumors, notably germ cell tumors, head and neck can- ing antibiotic yield [18]. Mannosylated glycans are com- cer and cervical carcinoma [3]. Although fermentation mon precursors of various glycoproteins and antibiotics medium optimization and manipulation of the pathway- including cell envelope polymers, polyenes, bleomycins specifc repressor blmR have been suggested to be use- and mannopeptimycins. Mannose can be sequentially ful for enhancing bleomycin production [4–6], the lower converted by phosphomannose isomerase (ManA), phos- yields and complex nutrient demands in rich medium phomannomutase (ManB), and GDP-mannose pyroph- have been major difculties in the industrial production osphorylase to form GDP-mannose. During bleomycin of bleomycins. biosynthesis, GDP-mannose and its isomer GDP-gulose Streptomyces species inhabit specifc environments are attached to the bleomycin aglycone through glyco- with changing nutrient availability, and their morpholog- sylation, which is responsible for the cellular recogni- ical development is tuned to coincide with the synthesis tion, uptake and metal ions chelation of bleomycins [19]. of secondary metabolites, including antibiotics. Under As the fnal step in the biosynthesis of most glycopep- starvation, multiple regulatory networks are employed by tide antibiotics, the genetic engineering of glycosylation Streptomyces to make a decision whether to continue veg- could not only result in hyper-susceptibility to antibiot- etative growth or form spores. Once it enters the spores ics and diverse derivatives of natural products, but its development, a series of genes are transcribed, which reaction efciency usually dictates the ultimate yield of activate the synthesis of antibiotics and morphological an antibiotic [20–23]. In addition to the glycosyl moie- diferentiation [7, 8]. Te onset of antibiotic synthesis not ties, the hybrid peptide-polyketide skeleton of bleomy- only depends on the expression of the specifc biosyn- cin is directly synthesized by nine common amino acids thetic gene clusters, but is also afected by the regulation [24], which indicate that the intracellular primary meta- of intracellular response mechanism of environmental bolic pathways of S. verticillus might also play a dominant status [9]. GlcNAc, the main component of chitin, is con- role in bleomycin biosynthesis. To further increase the sidered to be a signaling molecule with diferent efects yield of bleomycin, it is necessary to fully understand the on spore development and antibiotic yields in Streptomy- changes of global intracellular metabolites and identify ces under nutrient-rich/poor conditions [10, 11]. Actu- key modules that are associated with the synthesis of ble- ally, the regulation of the GntR/HutC family repressor omycins. Metabolic profling analysis is widely employed DasR has been found to be essential for the development for the identifcation of biomarkers and modules asso- and morphogenesis process, which controls the expres- ciated with target product synthesis. Tis approach has sion of genes involved in chitin/GlcNAc metabolism, the been used to increase the outputs of natural products development-related phosphoglucosyltransferase system and bioenergy [25, 26]. (PTS), stress response and nearly all the known antibiotic In this study, the signaling molecule GlcNAc greatly biosynthesis pathways [10, 12–15]. In vivo, DasR binds initiated the bleomycin biosynthesis in liquid medium. to a conserved element in the promoter region of its tar- To elucidate the GlcNAc induction mechanism, the get genes and represses their expression. However, the efects of the global transcription factor DasR on Glc- metabolites N-acetylglucosamine 6-phosphate (GlcNAc- NAc metabolism and bleomycin synthesis were investi- 6P) and glucosamine-6-phosphate (GlcN-6P) in GlcNAc gated by RT-qPCR and EMSAs. Te elevated expression catabolism would competitively bind to DasR, which is of the blmT gene under GlcNAc induction was found then released it from the target sites [14–16]. It has been to be the main cause of the improvement of bleomy- reported that the addition of GlcNAc to a niche simu- cin yield. To further increase bleomycin production lates a signal similar to cell wall lysis under nutritional under GlcNAc induction, gene blmT was overexpressed Chen et al. Microb Cell Fact (2020) 19:32 Page 3 of 17

and the supply of the precursor GDP-mannose was Gene identifcation involved in GlcNAc metabolism also enhanced by genetically engineering the mannose To extract genomic DNA used for chromosome walk- catabolism pathway. Assisted by comparative metabolic ing, the mature spores were suspended in Tryptic Soy profling analysis, the production of bleomycin A2 and Broth (TSB) medium (BD Biosciences, America) with B2 was respectively improved to 61.79 and 36.9 mg/L in the addition of 0.4% glycine and 10% sucrose (m/v). After the GlcNAc-derived chemically defned medium. 48 h of cultivation, 1 mL of culture was taken to collect the mycelium by centrifugation, which was then used to extract genomic DNA according to the protocol of the Materials and methods TIANamp DNA Kit (Tiangen, Beijing). To clone Strains, plasmids and bleomycins fermentation the conserved DNA fragments of genes related to Glc- Streptomyces verticillus ATCC 15003 purchased from NAc metabolism, a potential bleomycins producer Strep- American type culture collection (ATCC) was used tomyces mobaraensis DSM 40847 [28], which previously as the bleomycins-producing wild-type strain. All the was classifed as Streptoverticillium [29] and used as ref- strains and plasmids used in this study were listed in erence strain. Te amino acids and DNA sequences of Additional fle 1: Table S1. Te spores of S. verticillus gene dasR (Gene ID: WP004937563.1), manA (Gene ID: were preserved on ISP4 solid medium [27] with the WP004953455.1) and nagA (Gene ID: WP004954541.1) addition of 0.5 g/L yeast extract and 1 g/L tryptone in S. mobaraensis DSM 40847 were downloaded from the and minimal medium (MM) solid medium, respec- National Center for Biotechnology Information (NCBI) tively. MM solid medium contained 0.5 g/L l-aspara- and submitted to protein/nucleotide BLAST to fnd the corresponding target sequences with high homology in gine, 0.5 g/L K­ 2HPO4, 0.2 g/L MgSO­ 4·7H2O, 0.01 g/L Streptomyces. Ten, multiple sequence alignments were FeSO­ 4·7H2O and 6 g/L mannitol. For bleomycin pro- duction, the mature spores were cultured for 48 h in performed using Clustal Omega (https​://www.ebi.ac.uk/ seed medium containing 20 g/L soybean powder, 25 g/L Tools​/msa/clust​alo/) to identify highly conserved amino soluble starch, 5 g/L glucose, 1 g/L ­K2HPO4, 0.5 g/L acids/nucleotides regions, which were used for design- ing degenerate primers. Te PCR using genomic DNA ­ZnSO4, 0.1 g/L CuSO­ 4·5H2O and 2 g/L CaCO­ 3. Aliquots comprising 4 mL of the pre-culture were transferred of S. verticillus ATCC 15003 as template with degener- and cultivated for 7 days in complex fermentation ate primers were performed to amplify the conserved medium, modifed ISP4 liquid medium, medium I DNA fragments of dasR, manA and nagA. Based on the and medium II, respectively. Te complex fermenta- conserved DNA regions, the SiteFinding PCR was per- tion medium was the same as described in the previ- formed according to the published protocol with minor ous literature [4]. Te modifed ISP4 liquid medium modifcations [30, 31]. A detailed schematic diagram contained 20 g/L soluble starch, 20 g/L dextrin, 1 g/L was presented in Additional fle 1: Fig. S1. A 20 μL Site- Finding PCR mixture contained 10 μL 2 Phanta Max ­KH2PO4, 1 g/L ­MgSO4·7H2O, 1 g/L NaCl, 3 g/L ­KNO3, × Bufer, 0.5 μL dNTP Mix (10 mM each), 1 μL SiteFinder 0.001 g/L ­FeSO4·7H2O, 0.001 g/L ­MnCl2, 0.001 g/L (10 μM), 0.5 U Phanta Max Super-Fidelity DNA Poly- ­ZnSO4·7H2O and 5 g/L 3-Morpholinopropanesulfonic acid (MOPS). Medium I was formed by adding 5 g/L merase (Vazyme Biotech Co., Ltd, Nanjing, China) and of GlcNAc to modifed ISP4 liquid medium. Medium 50–100 ng template DNA. Te NotI site and six degen- II was composed of 10 g/L mannose, 5 g/L chitosan, erate nucleotides (NNNNNN) of SiteFinder-1 from Tan 20 g/L GlcNAc, 40 g/L dextrin, 1 g/L KH­ 2PO4, 1 g/L and colleagues’ protocol [31] were replaced by XbaI and NNNCGG, and the fnal SiteFinder (5′ CACGAC​ ACG​ ​ ­MgSO4·7H2O, 1 g/L NaCl, 6 g/L ­KNO3, 0.001 g/L CTA​CTC​AAC​ACA​CCA​CCT​CGC​ACA​GCG​TCC​TCA​ FeSO­ 4·7H2O, 0.001 g/L ­MnCl2, 0.001 g/L ­ZnSO4·7H2O and 5 g/L MOPS. Te pH of the seed medium and fer- AtctagaNNNCGG​GCGC 3′) in our study was used for mentation medium was adjusted to 6.5 with 3 mol/L the efcient amplifcation of high GC template DNA. NaOH solution. Escherichia coli DH5α and S17-1 were After the SiteFinding PCR, the frst nested PCR was cultured in Luria–Bertani medium (LB). employed to amplify the target DNA fragment. A 5 μL Due to the water-solubility characteristic of bleomy- mixture containing 2 μL of 10 μM SFP1, 1 μL of 10 μM cins, the fermentation broth of S. verticillus was centrifu- gene specifc primer 1 (GSP1) and 2 μL of 2× Phanta gated at 12000 r/min for 10 min, following by fltration Max Bufer was added to the 20 μL SiteFinding PCR through a 0.22 μm micropore membrane. Te superna- reaction mixture. Te largest DNA fragment among the tant was subjected to High Performance Liquid Chroma- amplifcation products of the frst nested PCR was used tography (HPLC) 1200 equipped with a Venusil MP-C18 as the template, the second round of nested PCR was performed and the mixture (50 μL) contained 25 μL column (5 μm, 250 mm × 4.6 mm). Te HPLC program was described in previous literature [4]. of 2× Phanta Max Bufer, 1 μL of dNTP Mix (10 mM Chen et al. Microb Cell Fact (2020) 19:32 Page 4 of 17

each), 2 μL of SFP2 (10 μM), 2 μL of GSP2 (10 μM), 1 using T4 DNA ligase (Termo Fisher Scientifc, USA) to U of Phanta Max Super-Fidelity DNA Polymerase and generate pIB139/manA/manB. Te PCR product of gene an appropriate volume of ­ddH2O. Te primers used for blmT was digested by XbaI and NotI, which was cloned the SiteFinding and nested PCR were presented in Addi- into pIB139/manA/manB to generate pIB139/BlmT/ tional fle 1: Table S2. To construct plasmids for sequenc- ManAB. After the verifcation by restriction enzyme ing, the amplifcation products from the second round digestion and sequencing, the correct recombinant plas- of nested PCR were purifed using FastPure Gel DNA mid was introduced into S. verticillus ATCC 15003 by Extraction Mini Kit (Vazyme Biotech Co., Ltd, Nan- conjugal transfer mediated by E. coli S17-1. Te overpro- jing, China). After double digestion with the restriction duction strain OBlmT/ManAB was selected via apramy- enzymes PstI and XbaI, the fragments were cloned into cin resistance screening and sequencing. Besides, strains the plasmid pUC18 and then transformed into E. coli DBlmR (BlmR deletion strain) and DBlmT-ΔBlmR (BlmR DH5α. Te corresponding plasmids were sequenced by deletion and BlmT overexpression strain) were con- Genewiz (Suzhou, China) and the sequences were depos- structed based on the protocol described in our previous ited into the GenBank. Te GenBank accession numbers literature [4]. of dasR-dasA, dasB-dasC-dasD-nagB, manA, manB and nagK-nagA were MN380024, MN380025, MN380026, RT‑qPCR MN380027 and MN380028, respectively. Te DNA To study the efect of GlcNAc induction on the expres- sequences were translated to corresponding amino acids sion of das genes and bleomycins synthesis genes, 4 mL sequences using the Bacterial, Archaeal and Plant Plas- of pre-culture of S. verticillus ATCC 15003 was inocu- tid Code (transl_table = 11) on NCBI (https​://www.ncbi. lated into the modifed ISP4 fermentation medium. After nlm.nih.gov/orf​nder/). the cultivation for 36 h, GlcNAc with a fnal concentra- tion of 5 g/L was added into the fermentation medium. Construction of gene engineering strains RNA samples were collected by centrifugation (4 °C For the overexpression of single gene, the whole nucleo- and 8000×g) at 0 h and 2 h after the GlcNAc addition. tide sequences of gene manA (GenBank: MN380026), RNA extraction and cDNA synthesis were performed as manB (GenBank: MN380027), blmC (GenBank: described in previous literature [4]. Te RT-qPCR was AAG02371.1), blmD (GenBank: AAG02370.1), blmE carried out using LightCycler­ ® 480 SYBR Green I Mas- (GenBank: AAG02369.1), blmF (GenBank: AAG02362.1), ter (Roche, Switzerland) in a LightCycler 480 (Roche, blmG (GenBank: AAG02361.1) and blmT (GenBank: Switzerland). Te primer pairs used in RT-qPCR were AAG02352.1) were amplifed by PCR using correspond- listed in Additional fle 1: Table S2. Te 20 μL qRT-PCR ing primer pairs (Additional fle 1: Table S2), and were reaction mixtures contained 2 μL of cDNA, 1 μL of for- respectively ligated into the integrative plasmid pIB139 ward/reverse primers, 10 μL of 2× Master Mix and 6 μL using the ClonExpress II One Step Cloning Kit (Vazyme, of PCR grade water. Te same volume (2 μL) of reverse Nanjing, China). Te recombinant plasmids were intro- transcription mixture containing a DNase I-treated duced into E. coli S17-1 for conjugal transfer into S. verti- RNA sample without reverse transcriptase was used as cillus ATCC 15003 according to the literature [32]. After negative control to exclude interference from residual screening for positive transformants, genomic DNA genomic DNA. Te 16S RNA was used as internal con- extraction and sequencing, the overexpression strains of trol, and the expression levels of target genes in medium manA, manB, blmC, blmD, blmE, blmF, blmG and blmT I were evaluated by normalizing them to those of S. verti- were constructed and designated as OManA, OManB, cillus cultured in modifed ISP4. Tree independent bio- OBlmC, OBlmD, OBlmE, OBlmF, OBlmG and OBlmT, logical replicates were conducted in our study. respectively. For the construction of bleomycin overproduction Protein purifcation and EMSAs strain, the encoding sequences of blmT, manA and manB For the production of 6 × His-tagged DasR, the DNA were amplifed using the corresponding primer pairs sequence of DasR without stop codon was amplifed listed in Additional fle 1: Table S2. Te PCR product of using primers PdasR/F and PdasR/R, in which the 6 × His manA was cut with NdeI and PstI, which was then cloned tag was introduced by PdasR/R. After the digestion by into pUC18 to generate pUC18/manA. After the diges- NdeI and BamHI, the fragment of dasR was cloned into tion with PstI and XbaI, the segment of manB was ligated plasmid pET-3c. Te obtained pET-3c/DasR was trans- with pUC18/manA to obtain the plasmid pUC18/manA/ formed into host E. coli BL21 Rosetta (DE3) for DasR manB. To construct the manAB overexpression plasmid, expression. Te E. coli BL21 Rosetta (DE3)/pET-3c/DasR the fusion fragment of manA and manB was digested by were cultivated at 37 °C for 2–3 h. After the optical den- NdeI and XbaI, which was then cloned into the pIB139 sity at 600 nm reached 0.6–0.7, a fnal concentration of Chen et al. Microb Cell Fact (2020) 19:32 Page 5 of 17

0.2 mM iso-propyl-β-d-thiogalactoside (IPTG) was spectra and the energy of ionization was 70 eV, the ioni- added to induce the production of DasR. Te purifcation zation temperature was 250 °C. Te MS scanning range of DasR was performed as described as previous litera- was 50–650 m/z. Both of the mass spectrum extraction ture [4] and SDS-PAGE profle was showed in Additional and identifcation of metabolites were performed by Agi- fle 1: Fig. S2. Besides, the 6 × His-tagged BlmR was also lent Masshunter Qualitative Analysis B.08.00. Te metab- purifed [4] and used to verify the efect of GlcN-6P on olites detected by GC–MS were shown in Additional the binding of BlmR to its target site. Te partial DNA fle 1: Table S3. After the normalization with the internal sequences spanning the potential dre sites in the pro- standard succinic d4 and cell biomass, the relative abun- moters of dasA, nagB and nagKA were acted as probes dance matrix of the whole identifed metabolites at each in EMSAs (Additional fle 1: Fig. S3), in which all the sin- sampling time point was loaded into SIGMA 13.0 (Umet- gle-strand DNA and its complementary fragments were rics, Umea, Sweden) for Principal Component Analysis chemically synthesized and labeled with Cy5 at 5′-end. (PCA) and Partial Least Squares Analysis (PLS). Five bio- After mixing equally in the annealing bufer, the double- logical replicates were conducted for each sample. stranded probes P­DasA, ­PNagB and P­NagKA were respec- tively obtained by touchdown PCR. Furthermore, the Results Bleomycin fermentation characteristics and morphological whole promoter probe ­PBlmR and BlmR binding sequence changes in S. verticillus mediated by GlcNAc probe ­PBlmR-1 were constructed according to Chen et al. [4], both were present in Additional fle 1: Fig. S3. DasR The yields of bleomycins A2 and B2 produced by S. ver- and various probes were incubated for 30 min in EMSAs ticillus ATCC 15003 in complex fermentation medium bufer, the mixture was then measured by 6% native reached 22.18 ± 3.29 mg/L and 38.26 ± 2.36 mg/L, polyacrylamide gels. To investigate whether GlcNAc respectively, while none were detected in modified metabolites could induce the dissociation of DasR-DNA ISP4 medium, even with the addition of 1 g/L tryptone and BlmR-DNA complexs, the diferent concentrations and 0.5 g/L yeast extract. To find the key factors affect- of GlcNAc and GlcN-6P were used as additives in the ing the synthesis of bleomycins, 5 g/L of glucose, man- EMSAs system. Besides, the reaction mixture without nose, fructose, arabinose, lactose, galactose, chitin, inducers was used as control group. GlcNAc and d-Glucosamine (GlcN) were individually added to the fermentation medium as supplementary Gas chromatograph mass spectroscopy (GC–MS) carbon sources. Surprisingly, only the addition of 5 g/L determination and statistical analysis mannose in modified ISP4 medium made the yield of To depict the intracellular metabolites profles response bleomycin reach the detection limit of HPLC, while to diferent fermentation conditions, 10 mL of the 5 g/L of GlcNAc in modified ISP4 (medium I) sig- OBlmT/ManAB fermentation broth from cultures in fer- nificantly increased the production of bleomycin A2 mentation medium I and II, respectively, was collected at and B2 to 22.91 ± 3.36 mg/L and 11.44 ± 2.39 mg/L, 48 h, 84 h and 108 h. Each 10 mL sample was quenched respectively (Fig. 1a). It was predicted that the carbo- immediately with 40 mL of 60% pre-cooled methanol hydrate metabolism might be one of the limiting fac- (− 80 °C, v/v) and centrifuged (6000×g, 4 °C) to collect tors for bleomycins overproduction. In S. coelicolor, cell pellet, which was then washed three times with 0.9% GlcNAc has been proven to be a signaling molecule NaCl solution (4 °C, w/v). Subsequently, the extraction related to the regulation of morphological differen- and chemical derivatization of intracellular metabolites tiation and the synthesis of antibiotic in response to were conducted according to the methods published by changes in the nutritional status [10]. Next, spores Xia et al. [25]. Succinic d4 (St. Louis, MO) was added in of S. verticillus ATCC 15003 were streaked onto ISP4 advance and served as the internal standard for GC–MS and MM solid medium with the addition of different analysis. All the derivatized samples were measured using concentrations of GlcNAc (Additional file 1: Fig. S4). a GC–MS using Agilent 7890B/7000D equipped with GlcNAc concentrations over 5 g/L led to develop- an HB-5 capillary column (30 m × 0.25 mm × 0.25 μm, mental retardation and the aerial hyphae did not form Agilent) for gas chromatography. Te parameters for grey-green spores, while colonies growing on ISP4 GC–MS determination were set as following: split ratio solid medium containing 25 g/L of GlcNAc exhibited was 5:1; both of injector temperature and interface tem- an abnormal, wrinkled phenotype. On the contrary, perature were set as 280 °C; the velocity of carrier gas the addition of 5 g/L GlcNAc in MM could accelerate (helium) was 1.0 mL/min. Te column temperature pro- spore maturation, but aerial mycelia were prevented gram encompassed 70 °C hold for 2 min, followed by a from developing into spores at high concentrations 5 °C/min ramp to 280 °C, where it was held for 3 min. (25 g/L GlcNAc). Overall, GlcNAc not only improved Te ionization mode was electron ionization (EI) mass the production of bleomycins but also induced a series Chen et al. Microb Cell Fact (2020) 19:32 Page 6 of 17

a b (GlcNAc)2~5 Bleomycin A2+ B2 GlcNAc DasA Medium I DasC DasB PT A2 Modified ISP4 +Man S NagK DasD GlcNAc-6P GlcNAc (GlcNAc)2~5 B2 Acetate NagA GlcN-6P NH3 NagB Fru-6P Glycolysis

ManA ManB BlmC BlmG 10 15 20 25 Man-6P Man-1P GDP-Man GDP-Gul Time (min)

c

dasR dasA dasB dasC dasD nagB 771 bp 1278 bp 978 bp 831 bp 1524 bp 783 bp

nagK nagA d 978 bp 1155 bp

dre sequence ACTGGTCTAGACCACT dasA ACTGGTCTA A A CCAT T nagB A T TGGTCTA T ACCAC C nagK A AAGGT C TAG T CCACT Fig. 1 Efect of GlcNAc addition on the fermentation characteristics of S. verticillus in chemically defned medium and schematic diagram of GlcNAc metabolism. a Bleomycin productions in modifed ISP4 with 5 g/L mannose and modifed ISP4 medium with 5 g/L GlcNAc (medium I). The HPLC spectrum of standard bleomycins was showed in cyan curve, and the peak times of bleomycin A2 and B2 had been marked by black dotted lines. b Metabolic pathway and enzymes in GlcNAc metabolism and GDP-mannose synthesis. c Length and organization of the gene sequences of the dasR, dasA, dasBCD, nagB and nagKA operons; the dre elements located in the promote region of dasA, nagB and nagK are indicated in red columns. d WebLogo of the dre element, and nucleotide sequence of dre motifs in S. verticillus. The DNA binding sequences of DasR were collected from literatures [10, 42], and were deposited to WebLogo to create sequence logos. The dre sequence was proposed by Rigali et al. [10]. Based on MAST analysis, the DNA sequences of the dre motif in the promoter region of dasA, nagB and nagK were shown, in which the red font indicated similarity with the dre consensus sequence

of morphological changes under different nutritional Regulation of GlcNAc metabolism by the global regulator conditions. Consequently, we decided to investigate DasR the effect of GlcNAc on bleomycin synthesis in S. ver- Since the whole genome sequence of S. verticillus ticillus in hopes of obtaining higher bleomycins titers. ATCC 15003 has not been published, the genes related Chen et al. Microb Cell Fact (2020) 19:32 Page 7 of 17

to GlcNAc metabolism (dasR, dasA, dasB, dasC, dasD, to the DasR-PNagB complex and the occurrence of a new nagK, nagA and nagB) in S. verticillus were identi- shifted band, while over 50 mM of GlcN-6P would lead to fed using SiteFinding PCR (Fig. 1b, c). Te amino acid the dissociation of the DasR-PNagKA complex but had no sequence of DasR showed 88.93% and 88.49% identity obvious impact on the DasR-PDasA complex (Fig. 2a, b). with its homologs from S. coelicolor A3(2) and S. griseus, To further verify whether the addition of GlcNAc in respectively. Te dasR was located adjacent to dasABCD the liquid medium relieved the inhibition of amino sugar operon, which is involved in the transport and metabo- metabolism by DasR, the fermentation characteristics of lism of N,Nʹ-diacetylchitobiose in S. coelicolor [33]. S. verticillus ATCC 15003 after GlcNAc addition at dif- Similarly, dasABCD in S. verticillus was also found to be ferent times were frst investigated. Te results indicated located upstream of dasR, and showed 69.88%, 77.37%, that the addition of GlcNAc during 0–48 h resulted in a 74.64% and 69.12% amino acid sequence identity with similar bleomycin yield (Additional fle 1: Fig. S5). Here, their respective homologs in S. coelicolor A3(2). Com- GlcNAc was added to the modifed ISP4 medium after pared with dasABCD, a divergently transcribed gene the cultivation of 36 h, at which time the strain was in nagB possessed higher amino acid identify (80.46%) with the early exponential phase. Samples for RT-qPCR analy- its homologs in S. coelicolor A3(2). Likely due to the pres- sis were collected at 0 h and 2 h after GlcNAc induction. ence of special sequence features, a partial intergenic Genes with relative expression exceeding 2 or below 0.5 region between dasA and dasB in S. verticillus could not were considered signifcantly diferentially expressed in be resolved successfully by Sanger sequencing. Addition- this study. As shown in Fig. 2c, the expression levels of ally, nagK and nagA in S. verticillus showed 71.33% and dasA, dasB, dasC and dasD did not change obviously, 72.32% identity with SCO4285 (nagK) and SCO4284 but that of dasR was increased at 2 h after induction (nagA) in S. coelicolor A3(2), respectively. Te amino acid with GlcNAc. Genes in the GlcNAc catabolism pathway sequence alignments indicated that the enzymes involved include nagK, nagA and nagB, with nagK and nagA shar- in GlcNAc catabolism in S. verticillus might present con- ing a single transcriptional unit (Fig. 1b, c). Te elevated siderable functional similarity to those of S. coelicolor expression levels of nagA and nagB at 2 h indicated that A3(2). GlcNAc induction inhibited the binding of DasR to the GlcNAc induction could relieve the repression of DasR dre sites in the promoter regions of the nagB, nagK and on the expression of nag and das genes in the amino nagA genes. sugar metabolism pathway, as well as the negative efect on pathway-specifc regulators in the antibiotic synthesis Efect of GlcNAc metabolism on bleomycin biosynthesis gene clusters [15]. DasR has been proved to bind to 16 bp Gene blmR, orf28, blmX and blmV are the frst genes in DasR-responsive elements (dre element) in the promoter operons I, II, III and IV of the bleomycin gene cluster, regions of its target genes. Moreover, the intermediate respectively. Te RT-qPCR results indicated that only the products of GlcNAc catabolism, especially GlcN-6P, can expression level of blmR was signifcantly increased, while induce the dissociation of the DasR-DNA complex by those of orf28, blmX and blmV were not afected in the competitively binding to DasR [14, 16]. Here, the known presence of GlcNAc (Fig. 2d). As an autoregulator, BlmR DasR binding sites identifed in Streptomyces were col- is previously characterized as a transcriptional repressor lected and loaded into WebLogo (http://weblo​go.berke​ of bleomycin production that binds to a 12-2-12 bp DNA ley.edu/logo.cgi) to create a sequence logo (Fig. 1d). fragment in its own promoter to inhibit the expression Tree DasR binding sites in the promoter regions of of the transcription unit composed of blmR and blmT dasA, nagK and nagB, with high degrees of similarity to [4]. According to gene annotation in NCBI, gene blmT the dre sequence, were respectively identifed in S. verti- encodes a putative transporter with a conserved sulfate cillus using the Motif Alignment & Search Tool (MAST) transporter and anti-sigma factor antagonist domain. (Fig. 1d). Te probes P­ DasA, ­PNagB and P­ NagKA contain- Te up-regulation of the transcription level of blmR had ing the corresponding dre sites in the promoter regions no direct inhibitory efect on the expression of blmT, of dasA, nagB and nagK were used in EMSAs, and the which increased nearly 2.5 times after GlcNAc induction results showed that DasR indeed had the ability to bind (Fig. 2c). No dre sites were found in the promoter of the to the promoter regions of dasA, nagB and nagK in S. blmR-blmT operon, and DasR could not bind to ­PBlmR verticillus. As an intracellular metabolite, high concen- in vitro, which was verifed by EMSAs (Fig. 2e). It was trations of GlcN-6P have been considered to be lethal. suggested that the change in the transcription level of the Tus, the efect of the GlcN-6P ligand on the binding of blmR-blmT operon was not caused by the disassociation DasR was also detected in vitro by EMSAs. Te addi- of the DasR-dre complex. Whether the GlcNAc metabo- tion of GlcN-6P (over 100 mM) into the EMSAs mixture lites directly afect the binding of BlmR to its target 12-2- resulted in the loss of the shifted bands corresponding 12 bp DNA sequence ­(PBlmR-1) was therefore an open Chen et al. Microb Cell Fact (2020) 19:32 Page 8 of 17

a His6-DasR(nM)0100200 500 0100 200500 0100 200500

PDasA ++ ++------PNagB -- --++ ++-- -- PNagKA ------++ ++

DasR-DNA complex

Free DNA

b His6-DasR(nM)0100100 100100 0100 100100 100 0 500 500500 500 GlcN-6P(mM)0050100 2000 050100 200 00 50 100200

PNagB + ++++------

PNagKA -- ---+++++ -- ---

PDasA ------++ +++

Blocking bands

Free DNA

c d 6 3.0 0h 0h 2h 5 2.5 2h

s 4 2.0 ld s fo

3 fold 1.5 ange Ch

2 Change 1.0

1 0.5

0 0.0 dasR dasA dasB dasCdasDnagAnagB blmX blmV orf28 blmR blmT e f

His6-BlmR(nM)0100100 100100 100 His -DasR(nM )0100200 500 6 GlcNAc(mM) 00100200 00 PBlmR ++ ++ GlcN-6P(mM) 00 00100200

PBlmR-1 ++ + +++

BlmR-DNA complex

Free DNA Free DNA

Fig. 2 Efect of DasR on the promoter ability of das genes, nag genes and blmR. a EMSAs of His­ 6-DasR binding to probes ­PDasA, ­PNagB and ­PNagKA. b EMSAs analysis of the efect of GlcN-6P on the binding of DasR to probes P­ DasA, ­PNagB and ­PNagKA. c Change folds of GlcNAc metabolism genes at 0 h and 2 h after GlcNAc induction. d Transcription levels of bleomycin biosynthetic gene cluster at 0 h and 2 h after GlcNAc induction. e EMSAs

displayed the binding of ­His6-DasR to the whole promoter region of gene blmR. f EMSAs analysis indicating the efect of GlcNAc and GlcN-6P on the BlmR-DNA complex Chen et al. Microb Cell Fact (2020) 19:32 Page 9 of 17

question. Te result from EMSAs showed that neither a 2.5 GlcNAc nor GlcN-6P could induce the dissociation of 0 h 2 h the BlmR-DNA complex (Fig. 2f). Due to the positive role 2.0 of the elevated expression of BlmT in bleomycin biosyn- thesis, the strain OBlmT was constructed, and the titers 1.5 of bleomycin A2 and B2 were respectively increased to 1.0 32.72 ± 1.83 mg/L and 20.18 ± 3.09 mg/L in the presence Change folds of GlcNAc, which were close to those of the BlmR dele- 0.5 tion and BlmT overexpression strain DBlmT-ΔBlmR. In addition, the titers of bleomycin A2 and B2 of the BlmR 0.0 deletion strain DBlmR in modifed ISP4 medium without manA manB blmC blmD blmE blmF blmG GlcNAc addition were respectively 18.04 and 11.92 mg/L, b 40 BLM-A2 which were lower than with the addition of GlcNAc 35 BLM-B2 (23.88 and 14.82, respectively). It could be inferred that 30 the global regulatory efect of GlcNAc on bleomycin syn- 25 thesis was not limited to the relief of negative regulation 20 of BlmT by the pathway-specifc regulator BlmR, but was related to the other factors as well. Nevertheless, genetic 15 engineering of blmT under GlcNAc induction ofered a 10 Bleomycin yield (mg/L) feasible method for increasing the bleomycin yield. 5 0

pe A B C D E F G Improved GDP‑mannose supply to increase bleomycin an an lm lm lm lm lm d-ty M M B B B B il O O O O OB O O yield under GlcNAc induction W According to the pathway analysis of amino sugar metab- Fig. 3 a Efect of GlcNAc induction on the expression of genes related to GDP-mannose synthesis and glycosylation. b Efects of the olism, GlcNAc can be converted to fructose-6-phosphate overexpression of genes in mannose catabolism on the production (frucose-6P) catalyzed by NagA and NagB. Improved of bleomycin A2 and B2. The data were the average values from three transcription levels of nagA and nagB under GlcNAc repeated fermentation trials addition indicated that GlcNAc might be assimilated efciently by S. verticillus. Fructose-6P is not only the end-product of GlcNAc metabolism, but it can also be converted into GDP-mannose and GDP-gulose in bleo- bleomycins glycosylation were overexpressed in strains mycin biosynthesis, catalyzed by ManA, ManB, the sugar OManA, OManB, OBlmC, OBlmD, OBlmE, OBlmF synthase BlmC and the sugar epimerase BlmG (Fig. 1b). and OBlmG, respectively. Te fermentation results in GDP-mannose and GDP-gulose are the direct precursors Fig. 3b showed that the total production of bleomycin A2 of the disaccharide unit that are attached to the bleomy- and B2 was higher in OManA and OManB than in the cin aglycone through glycosylation catalyzed by the sugar other strains, with bleomycin A2 and B2 titers reach- transferase BlmE, hydroxylase BlmF and carbamoyl- ing 31.80 ± 1.93 mg/L and 19.63 ± 2.84 mg/L in OManA transferase BlmD [32]. Whether the increased bleomy- and 25.3 ± 1.42 mg/L and 21.9 ± 2.05 mg/L in OManB, cins yield under GlcNAc induction could be associated respectively. Tese results indicated that ManA and with enhanced biosynthesis of GDP-mannose, the DNA ManB might catalyze rate-limiting steps that were critical fragments of manA and manB from S. verticillus were for increasing bleomycin production. For the improve- sequenced by SiteFinding PCR, and the corresponding ment of bleomycin yield under GlcNAc induction, the amino acid sequences exhibited 69.10% and 82.09% iden- tandem overexpression strain OBlmT/ManAB was con- tity with SCO3025 (manA) and SCO3028 (manB) from structed, and its corresponding titers of bleomycin A2 S. coelicolor A3(2), respectively. RT-qPCR was utilized and B2 in medium I were increased to 40.37 ± 2.04 mg/L to measure the expression of genes involved in disaccha- and 23.51 ± 3.18 mg/L, respectively. ride unit synthesis (manA, manB, blmC and blmG) and glycosylation (blmE, blmF and blmD) in the presence of Identifcation of key modules related to bleomycin GlcNAc. Te results (Fig. 3a) indicated that the expres- overproduction based on diferent GlcNAc‑derived media sion of genes listed above was not obviously changed In addition to the efect of GlcNAc metabolism on (change folds below 2). To improve the specifc sugar transcription factors, a previous study indicated that precursor supply under GlcNAc induction, gene manA, GlcNAc assimilation could lead to changes in the con- manB, blmC, blmD, blmE, blmF and blmG related to centrations of various intracellular nitro-containing Chen et al. Microb Cell Fact (2020) 19:32 Page 10 of 17

metabolites (especially amino acids, purines, and pyri- Fig. 4a, fve parallel samples taken at the same time midines) [34]. On the basis of medium I, the optimiza- point were tightly clustered, and six samples were tion of the fermentation medium for the production of clearly separated using Hotelling’s T­ 2 test (95%), both bleomycins was performed using OBlmT/ManAB as of these results refected diferent intracellular meta- bleomycins-producing strain. Te obtained medium bolic characteristics in the selected fermentation II contained high levels of chitin-derived substrates phases. To connect the changes of metabolite fuxes to including chitosan and GlcNAc, which increased the bleomycins synthesis, PLS analysis of all the samples titers of bleomycin A2 and B2 to 52.16 ± 1.93 mg/L was carried out using metabolites as X variables and and 30.02 ± 3.04 mg/L, respectively. To investigate the bleomycin yield as the Y variable. In the PLS-derived key factors limiting the yield of bleomycins in GlcNAc- score plot (R2X(cum) = 0.745, R2Y(cum) = 0.934, derived media, the intracellular metabolic profles of Q2(cum) = 0.897) shown in Fig. 4b, the samples from strain OBlmT/ManAB in medium I and medium II diferent media were distributed in diferent quadrants, were assessed by comparative metabolomics. A total with samples from medium I being found in lower of 92 metabolites were detected, 72 of which were regions of quadrants than those from medium II. Simul- identifed by GC–MS (Additional fle 1: Table S3). taneously, the variable importance of the projection Subsequently, PCA was utilized to analyze the difer- (VIP) plots of each metabolite was generated, where a ences between the samples. From the PCA-derived higher VIP value (exceeding 1) indicated a more sig- score plot (R2X(cum) = 0.889, Q2(cum) = 0.809) in nifcant contribution to the diferences in bleomycin

a Coloredaccording to ObsID(Obs. Sec. ID:1) b M1-48 M1-84 M1-108 M2-48 M2-84 M2-108 M1-48 M1-84 M1-108 10 8 M2-48 M2-84 M2-108

5 4

0 0 t[2] t[2]

-5 -4

-10 -8

-15-1-0 50 510 -8 -4 048 t[1] t[1] c Histidine 1,3-propanediol GlcNAc Uridine Linoleic acid Leucine Malic acid Pyruvic acid Cysteine PGAL Tyramine Indole Stearic acid Inosine Phenylalanine Monopalmitin Quinolinate Metabolites Benzoic acid Oxalic acid Urea Adenine GMS 2-Oxoglutarate Glycerol Palmitic acid Methionine

0 1 2 VIP [1] Fig. 4 Statistical analysis of intracellular metabolites data measured by GC–MS. Samples were collected at 48 h, 84 h and 108 h for a comparison between medium I and medium II. a PCA- derived loading plots. b PLS-derived loading plots. Samples from medium I and medium II were inside the black and orange dashed ellipses, respectively. c PLS-derived VIP plots with 26 metabolites with VIP values exceeding 1. GlcNAc, N-acetylglucosamine; PGAL, glyceraldehydes 3-phosphate; GMS, glycerol monostearate Chen et al. Microb Cell Fact (2020) 19:32 Page 11 of 17

production. According to the VIP scores (Additional Further improvement of bleomycin yield by metabolic fle 1: Table S3) and KEGG pathway analysis, 30 profling analysis chemical substances were identifed as key metabo- As seen in Fig. 4c, 9 metabolites associated with amino lites related with bleomycins overproduction, most of acid metabolism were identifed as biomarkers related which were involved in the metabolism of amino acids, to bleomycin biosynthesis. Tose were histidine, leu- long-chain fatty acids, pyruvate and the TCA cycle. cine, cysteine, tyramine, indole, phenylalanine, qui- Te bleomycins aglycone is formed by the dehydration nolinate, benzoic acid and methionine. Among them, condensation of 9 amino acids, including one l-serine, histidine and cysteine are direct precursors of bleomy- two l-asparagines, one l-histidine, one l-alanine, one cins, while methionine can be converted into S-aden- l-threonine, one β-alanine and two l-cysteines [2]. osylmethionine (SAM) to provide methyl groups for Together, the biomarkers of amino acids metabolism bleomycins synthesis. Te changes of relative abun- can therefore be expected to play critical roles in the dance of these biomarkers are shown in Fig. 5a. Com- metabolic fux distribution and the supply of various pared with medium I, the relative abundance of amino acid precursors between chitin-derived media. histidine increased with time and was always higher

a Medium I Medium II 2 15 2.4 Histidine Cysteine Leucine

10 1.6 1 e 5 0.8

0 0 0.0 48h84h 108h 48h84h 108h 48h 84h108h 5 Benzoic acid 6 Indole 6 Quinolinate

Relative abundanc 4

3 4 4

2 2 2 1

0 0 0 48h 84h 108h 48h84h 108h 48h84h 108h Time (h) b c 40 His Met Asn Tyr His Met Leu Phe 60 Trp Cys Asn Tyr Trp Cys Leu Phe 35

50 30

40 25

20 30 Bleomycin A2 (mg/L)

Bleomycin B2 (mg/L) 15 20 0122436486072 0122436486072 Feeding time (h) Feeding time (h) Fig. 5 a Comparative analysis of biomarkers associated with bleomycin production between medium I and medium II at 48 h, 84 h and 108 h. b, c Efects of exogenous addition of key amino acids in medium II on bleomycin A2 (b) and B2 (c) production at various times. The black dashed lines represented the productions of bleomycin A2 and B2 in medium II, which was used as the control Chen et al. Microb Cell Fact (2020) 19:32 Page 12 of 17

in medium II, while the relative abundance of cysteine Discussion in medium II was noticeably increased only at 108 h. Te synthesis of antibiotics in Streptomyces is infu- Tese results indicated that the fux of histidine and enced by multiple factors, such as primary metabolism, cysteine was enhanced in the later phase of bleomycins transcriptional regulation, nutritional status and mor- fermentation. Surprisingly, leucine was also identifed phological development [8]. To explore the limiting as a biomarker, although it does not directly contrib- factors during the synthesis of bleomycins, the optimi- ute to the supply of precursors for bleomycins syn- zation of the fermentation medium was frst conducted thesis. However, leucine can be converted into acetyl in modifed ISP4 liquid fermentation medium, in which CoA, which can be converted into the bleomycins pre- the strain was initially unable to synthesize bleomycins. cursor malonyl-CoA by acetyl-CoA carboxylases [35], Mannose is the direct precursor of GDP-mannose and and the relative abundance of leucine in medium II GDP-gulose, which together constitute the disaccharide was obviously higher than that in medium I. Phenyla- structure of bleomycins [36]. Te addition of mannose lanine, tyramine, indole, quinolinate and benzoic acid in modifed ISP4 liquid medium was able to improve the were associated with aromatic amino acids metabo- bleomycins production to the detection limit of HPLC, lism according to KEGG pathway analysis. In medium while a higher yield was achieved in the case of GlcNAc II, only the fux of benzoic acid was increased, while addition. In S. coelicolor A3(2), the high concentration phenylalanine and tyramine were not detected and the of GlcNAc blocks spore development and antibiotic relative abundance of indole and quinolinate gradu- production under nutrient-rich condition (R2YE solid ally decreased. It can be inferred that the weakening of medium), but it accelerates the sporulation and synthesis aromatic amino acids pathways appears to afect bleo- of the antibiotics undecylprodigiosin and actinorhodin mycin synthesis although none of them are the direct under nutrient-limited conditions (MM medium) [10]. precursors. Indeed, GlcNAc was found to inhibit the spore matura- To verify the deduced regulation points in bleomycins tion of S. verticillus in modifed ISP4 solid medium, but synthesis, a rational feeding experiment was conducted the production of bleomycins A2 and B2 was improved based on the above-described predictions. 1.5 g/L histi- following the addition of GlcNAc in modifed ISP4 liquid dine, leucine, cysteine, methionine, tryptophan, tyros- medium. It could be concluded that the efect of GlcNAc ine and phenylalanine were added into the medium II metabolism on bleomycins production in S. verticil- at diferent fermentation phases, respectively. As seen lus might be diferent from its efect on the induction of in Fig. 5b, c, the productions of bleomycin A2 and antibiotic synthesis in S. coelicolor under nutrient-limited B2 increased after the addition of histidine (36 h and conditions. 48 h), leucine (48 h) and cysteine (48 h and 36 h) in Several lines of evidence indicate that GlcNAc plays a medium II, respectively. Among the aromatic amino vital role in the control of morphological diferentiation acids, only tyrosine (48–60 h) enhanced the produc- and the onset of antibiotic synthesis based on the nutri- tion of bleomycin B2, while exogenous feeding of either tional status in the environment [11]. Te GntR family tryptophan or phenylalanine had the negative efect on repressor DasR is widely considered as a master regula- the bleomycin yields, which indicated that the down- tor that binds to specifc DNA sequences in the promoter regulation of the fux of aromatic amino acids during regions of genes involved in chitin/GlcNAc catabolism, fermentation would be benefcial for the accumula- sugar hydrolysis, antibiotic production and develop- tion of bleomycins. Although SAM is the precursor for mental regulation. However, the intermediate products bleomycin biosynthesis, the addition of methionine did of GlcNAc catabolism can induce the release of DasR not signifcantly promote the synthesis of bleomycins. from the target sites, and thereby trigger spore develop- Among the 9 amino acid precursors of the bleomycins ment and antibiotic synthesis [37]. In fact, a number of aglycone, asparagine was the only one that was not EMSAs studies have proven that DasR binds to a 16 bp detected in either medium. Te fermentation with the dre element and this binding is relieved by the GlcNAc addition of 1.5 g/L asparagine in medium II indicated catabolites GlcNAc-6P or GlcN-6P [16]. Te predic- that asparagine played an active role in bleomycins pro- tion of dre elements in Streptomyces is considered as duction based on the results of feeding at 36 h. To fur- a potential strategy for identifying the physiological ther improve the bleomycins production, the combined activities that are modulated by GlcNAc [12]. Tree dre feeding of asparagine (36 h), histidine (36 h), leucine sequence motifs in S. verticillus were identifed in the (48 h), and cysteine (36 h) in chitin-derived medium promoter regions of dasA, nagK and nagB, respectively. II was tested, and was found to increase the produc- According to the EMSAs results, the promoter activi- tion of bleomycin A2 and B2 to 61.79 ± 3.17 mg/L and ties of dasA, nagKA and nagB were indeed repressed by 36.9 ± 2.33 mg/L, respectively. DasR. However, high concentration of GlcN-6P could Chen et al. Microb Cell Fact (2020) 19:32 Page 13 of 17

alter the binding mode of DasR-PNagB and a new shifted transporters in nutrient sensing and the onset of develop- band appeared, indicating that DasR from S. verticillus ment are not been well understood. also possessed a similar mode of allosteric modifcation As a member of the ArsR/SmtB family regulators, to its orthologs NagR and ­DasRSerry, in which the DNA- BlmR has been verifed as an autoregulator that represses binding domain of NagR had been verifed to be re-ori- the expression of the transcription unit blmR-blmT by ented upon ligand binding [38, 39]. In this study, GlcNAc binding to its own promoter region [4]. Under the induc- induction not only relieved the inhibition of the expres- tion with GlcNAc, the increased expression levels of sion of genes related to GlcNAc catabolism by DasR, but blmR and blmT indicated that the binding of BlmR to its also overcame the efect of the increased expression of target DNA site might be changed or dissociated, leading DasR. Tis phenomenon has been perviously observed in to the relief of the negative regulation by BlmR. In fact, the wild-type strain S. coelicolor A3(2) in the presence of the binding of most ArsR/SmtB family transcriptional chitin (polymer of GlcNAc) [13]. Te stable expression of regulators to their target DNA sites is allosterically regu- dasABCD was consistent with the idea that the expres- lated under the control of various physiological factors sion of dasABCD controlled by DasR was induced by such as metal ions or oxidative signals [41]. However, the (GlcNAc)2~5 metabolism and not by GlcNAc metabolism binding of BlmR could not be afected by metal ions and [40]. oxidative signals [4]. Our study also indicated that both Tere was no doubt that the enhancement of GlcNAc GlcNAc and GlcN-6P could not act as ligands to induce metabolism in S. verticillus was ascribed to the removal the dissociation of the BlmR-DNA complex. GlcNAc is of the inhibition by DasR. It has been reported that the an important signaling molecule related to morphologi- biosynthesis of most antibiotics in S. coelicolor is initi- cal diferentiation in Streptomyces, where it can induce ated under GlcNAc induction in poor conditions, which changes of global metabolites and regulatory networks. is in fact achieved by impeding the binding of DasR to Te dynamic transcriptional regulation mechanism of the dre element in the promoter region of pathway-specifc repressor BlmR triggered by GlcNAc might be linked to regulators [14, 16]. Among them, DmdR1 is the only the complex regulatory cascades or metabolite spectrum. known repressor, which is responsible for the biosynthe- Nevertheless, it was clear that GlcNAc could improve the sis of the siderophores coelichelin and desferrioxamine. bleomycins yield by relieving the transcriptional repres- DasR could reduce the transcription of gene dmdR1 by sion of the blmR-blmT operon, and the enhancement of binding to the dre element located in its promoter region, BlmT expression under GlcNAc induction could ofer which improves the production of siderophores. Under a practical approach for the improvement of bleomycin the induction with GlcNAc, the expression of DmdR1 production. is initiated, which in turn represses the biosynthesis of According to the KEGG database, the end-product of siderophores [12]. In the bleomycin biosynthesis gene GlcNAc metabolism, fructose-6P, can be converted to cluster, BlmR acts as an ArsR family repressor that binds mannose-1P which is the direct precursor of the disac- to its own promoter to attenuate the transcription of the charide moiety of bleomycins. Te results of RT-qPCR blmR-blmT operon, and the combination of the deletion indicated that the transcription of genes involved in of blmR and overexpression of blmT could remarkably GDP-mannose synthesis and bleomycin glycosylation increase the bleomycin yield [4]. Whether DasR con- did not change obviously even when GlcNAc catabo- trols the transcription of BlmR through a similar regula- lism was enhanced. Consequently, the manA, manB, tion mechanism to that of dmdR1, no dre element was blmC, blmD, blmE, blmF and blmG genes were indi- found and EMSAs indicated that DasR could not bind vidually overexpressed to determine the limiting step in to the promoter region of BlmR in vitro. In addition to the specifc sugar precursor supply for bleomycins syn- the dre element, the binding of DasR to non-canoni- thesis. Fermentation experiments suggested that bleo- cal sites is also possible, but it requires the cooperative mycin yields of OManA and OManB were higher than interaction with other cofactors or changes of the DNA other strains, which suggested that ManA and ManB conformation, which cannot be observed from in vitro were the key enzymes controlling the fow of carbon experiment such as EMSAs [15]. Whether DasR afects toward the bleomycins disaccharide structure. Previ- the regulatory role of the repressor BlmR by binding to ous study found that the enzyme activity of ManA in S. the non-canonical sites is a subject of ongoing research coelicolor was lower than that in other organisms [42], in our lab. In addition, GlcNAc and its various polymers, and the carbon fux toward mannose catabolism might which are important signaling molecules in Streptomyces, hence be relatively elevated by the overexpression of are assimilated through several complex systems such as ManA. ManB is a bifunctional enzyme with phospho- PTS, DasABCD and NgcEFG, to cope with the shifting mannomutase and phosphoglucomutase activities, but nutrients conditions [11]. So far, the precise roles of these it is likely to give priority to the GDP-mannose synthesis Chen et al. Microb Cell Fact (2020) 19:32 Page 14 of 17

pathway in S. coelicolor [43]. Te high sequence identity to bleomycin biosynthesis in GlcNAc-derived media, (82.08%) of ManB from S. verticillus to its homolog from the intracellular metabolite profling was investigated by S. coelicolor might also indicate their large functional comparative metabolomics in this study. Here, histidine, similarity. Under GlcNAc induction, the combined over- cysteine, leucine, aromatic amino acids and methionine expression of blmT, manA and manB led to the increase were considered to present a high correlation with bleo- of the production of bleomycin A2 and B2 in medium I to mycins overproduction according to PLS analysis. It is 40.37 mg/L and 23.51 mg/L, respectively. known that methionine can be catalyzed to form SAM, Bleomycin aglycone is directly come from eight natu- which is an important methyl donor for antibiotic syn- ral amino acids and one unnatural amino acid β-alanine thesis and DNA methylation [44]. Tere are two meth- (Fig. 6). Moreover, GlcNAc assimilation has the posi- ylation reactions with the participation of SAM during tive efect on the abundance of intracellular amino acid the bleomycins synthesis, but the addition of methio- pools [34]. To explore the limiting metabolites related nine in medium II could not improve the production of

Chitosan

D-glucosaminide Glucose D-glusamine

Gluconolactone Gluconate Glucose 6-P Glusamine 6-P 6-P 6-P EMP PP

D-Mannose Frutose 6-P Ribulose 5-P Ribose 5-P PRPP Histidine

Mannose 6-P Frutose 1,6-P2 Erythrose Sedoheptulose 4-P 7-P Mannose 1-P Tyrosine Glycerate 1,3-P2 Shikimate Phenylalanine GDP-D-mannose Tryptophan

GDP-L-gulose Glycerate 3-P Serine Cystetine Homocystetine Methionine

Leucine PEP Glycine Valine Pyruvate Alanine AdoMet Isoleucine Threonine -Alanine Acetyl-CoA Malonyl-CoA NH3CO2 Lysine Aspartate Oxaloacetate Citrate Citruline Glutamine Asparagine Malate Isocitrate Carbamonyl-P TCA Fumarate AKG Glutamate Ornithine Arginine Proline Succinate Succinyl-CoA

Serine Histidine Alanine Cystetine Malonyl- AdoMet GDP-D-mannose CoA Asparagine Tyrosine -Alanine GDP-L-gulose

Bleomycins

Fig. 6 Schematic view of the metabolic pathway associated with bleomycin synthesis in S. verticillus. The precursors for bleomycin production were represented by colored rectangular boxes. The dashed arrows indicated that there were multiple reaction steps in this line. EMP, Embden– Meyerhof–Parnas pathway; PP, Pentose Phosphate pathway; PEP, Phosphoenolpyruvate; PRPP, 5-Phosphoribosyl 1-Pyrophosphate; TCA, Tricarboxylic acid cycle; AKG, α-Ketoglutaric acid Chen et al. Microb Cell Fact (2020) 19:32 Page 15 of 17

bleomycins. A similar lack of a promoting efect was also drug formulation. Here, signal molecule GlcNAc regu- reported in the synthesis of antibiotics carrying methyl or lation and assisted metabolic profle analysis efectively methoxy groups, which might result from the repression enhanced the bleomycin yield. Moreover, the utiliza- of methyltransferases by methionine [45]. As the direct tion of various chitin-derived constituents in medium precursors of the bleomycins aglycone, the addition of not only improved the applicability and economic fea- either histidine or cysteine could increase the bleomycin sibility of the fermentation process for industrial bleo- yield. Furthermore, an elevated bleomycins yield was also mycin production, but also ofered an example for the obtained via the addition of leucine. It has been proven other improving the bio-production processes of other that the addition of leucine can lead to the increase of antibiotics. intracellular pool of acetyl-CoA, which in turn could be easily transformed into malonyl-CoA through a one step Supplementary information enzymatic reaction [25]. Based on the stereochemical Supplementary information accompanies this paper at https​://doi. org/10.1186/s1293​4-020-01301​-8. structure of bleomycins, malonyl-CoA is the sole sub- strate used to assemble to the skeleton of the bleomycins Additional fle 1: Table S1. strains and plasmids used in this study. aglycone by the specifc hybrid NRPS-PKS synthases [46]. Table S2. Primers used in this study. Table S3. List of intracellular With the exception of tyrosine, the feeding of aromatic metabolites detected by GC–MS. Figure S1. Detail schematic diagram amino acids was not conducive to the increase of bleo- of chromosome walking by SiteFinding PCR and nested PCR. Figure S2. SDS-PAGE profle of 6 His-DasR used in EMSA assay. Figure S3. DNA mycin yield. According to the changes of relative abun- sequence of probes used× in EMSAs. Figure S4. Efect of GlcNAc addition dances of metabolites in aromatic amino acids pathway, on spore morphogenesis in diferent mediums. Figure S5. Fermentation it could be suggested that the intracellular metabolic fux characteristics of bleomycins in the diferent GlcNAc addition times. The fowing to aromatic amino acids might tend to decline control group was without GlcNAc. under bleomycins overproduction condition. Although asparagine was not detected in the fermentation process, Abbreviations GlcNAc: N-acetylglucosamine; RT-qPCR: Real time fuorescence quantitative its positive efect on the enhancement of bleomycins was PCR; EMSAs: Electrophoretic mobility shift assays; ManA: Phosphomannose verifed by feeding assay. Finally, the combined feeding of isomerase; ManB: Phosphomannomutase; PTS: Phosphoglucosyltransferase the four amino acids asparagine, leucine, histidine and system; GlcNAc-6P: N-acetylglucosamine 6-phosphate; GlcN-6P: Glucosamine- 6-phosphate; ATCC​: American type culture collection; MOPS: 3-Morpholino- cysteine in medium II further increased the production propanesulfonic acid; LB: Luria–Bertani medium; HPLC: High performance of bleomycins, illustrating the applicability of metabo- liquid chromatography; TSB: Tryptic Soy Broth medium; MM: Minimal medium; lomics in microbial bio-manufacturing. NCBI: National Center for Biotechnology Information; GSP: Gene specifc primer; IPTG: Iso-propyl-β-d-thiogalactoside; GC–MS: Gas chromatograph mass spectroscopy; EI: Electron ionization; PCA: Principal component analysis; Conclusions PLS: Partial least squares analysis; GlcN: d-Glucosamine; dre: DasR-responsive GlcNAc is a global signaling molecule and was demon- elements; MAST: Motif alignment and search tool; VIP: Variable importance of the projection; SAM: S-adenosylmethionine. strated in this study to signifcantly initiate the biosyn- thesis of bleomycins. To explore the efects of GlcNAc Acknowledgements on bleomycin biosynthesis, the das and nag genes We want thank Dr. Junhua Wang and Cheng Wang for the comments on our manuscript. involved in GlcNAc regulation and metabolism in S. ver- ticillus were investigated, and the results showed that Authors’ contributions the repression of the expression of nagB and nagKA by HC carried out the experimental work, analyzed the data and wrote the manuscript. JC performed the GC–MS assay and the data analysis, participated the global transcription factor DasR was relieved under in the manuscript editing and revise. PW and XW helped to partial experiment the induction with GlcNAc. Although no direct evi- and fgure processing. JPW was responsible for the experiment design and dence proved that the expression of BlmR was regulated supervision. All authors read and approved the fnal manuscript. by DasR, the negative efect of BlmR on its target gene Funding blmT was indeed relieved under GlcNAc induction, This work was fnancially supported by the National Key Research and Devel- which was the main cause of elevated bleomycin produc- opment program [No. 2018YFA0902200], and the National Natural Science Foundation of China [No. 21676189]. tion and ofered an efective strategy for strain improve- ment. By applying this strategy in combination with the Availability of data and supporting materials key rate-limiting enzymes ManA and ManB in bleomy- All data generated and analyzed during this study are included in this manu- script and in its Additional fle 1. cins glycosylation, the overproduction strain OBlmT/ ManAB was constructed. Assisted by metabolic profl- Ethics approval and consent to participate ing analysis, the production of bleomycin A2 and B2 in This article does not contain any studies with human participants or animals performed by any of the authors. GlcNAc-containing medium was ultimately increased to 61.79 and 36.9 mg/L, respectively, and the product ratio Consent for publication of 2:1 was close to the commercial bleomycin-based All authors consent to publish this manuscript. Chen et al. Microb Cell Fact (2020) 19:32 Page 16 of 17

Competing interests N-acetylglucosamine sensing and utilization in Streptomyces coelicolor The authors declare that they have no competing interests. and is subject to multi-level control. Mol Microbiol. 2010;75:1133–44. 18. Demain AL, Sanchez S. Microbial drug discovery: 80 years of progress. J Author details Antibiot. 2009;62:5. 1 Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin 19. Galm U, Hager MH, Van Lanen SG, Ju J, Thorson JS, Shen B. Antitu- University, Tianjin 300072, People’s Republic of China. 2 SynBio Research Plat- mor antibiotics: bleomycin, enediynes, and mitomycin. Chem Rev. form, Collaborative Innovation Center of Chemical Science and Engineering 2005;105:739–58. (Tianjin), School of Chemical Engineering and Technology, Tianjin University, 20. Zhou X, Wu H, Li Z, Zhou X, Bai L, Deng Z. Over-expression of UDP-glu- Tianjin 300072, People’s Republic of China. cose pyrophosphorylase increases validamycin A but decreases validoxy- lamine A production in Streptomyces hygroscopicus var. jinggangensis Received: 17 December 2019 Accepted: 5 February 2020 5008. Metab Eng. 2011;13:768–76. 21. Howlett R, Anttonen K, Read N, Smith MC. Disruption of the GDP- mannose synthesis pathway in Streptomyces coelicolor results in antibiotic hyper-susceptible phenotypes. Microbiology. 2018;164:614. 22. Lochlainn LN, Cafrey P. Phosphomannose isomerase and phospho- References mannomutase gene disruptions in Streptomyces nodosus: impact on 1. Umezawa H, Maeda K, Takeuchi T, Okami Y. New antibiotics, bleomycin A amphotericin biosynthesis and implications for glycosylation engineer- and B. J Antibiot. 1966;19:200–9. ing. Metab Eng. 2009;11:40–7. 2. Shen B, Du L, Sanchez C, Chen M, Edwards DJ. Bleomycin biosynthesis 23. Salcedo RG, Olano C, Fernández R, Braña AF, Méndez C, de la Calle F, Salas in Streptomyces verticillus ATCC15003: a model of hybrid peptide and JA. Elucidation of the glycosylation steps during biosynthesis of antitu- polyketide biosynthesis. Bioorg Chem. 1999;27:155–71. mor macrolides PM100117 and PM100118 and engineering for novel 3. Huang S-X, Feng Z, Wang L, Galm U, Wendt-Pienkowski E, Yang D, Tao derivatives. Microb Cell Fact. 2016;15:187. M, Coughlin JM, Duan Y, Shen B. A designer bleomycin with signifcantly 24. Du L, Sánchez C, Chen M, Edwards DJ, Shen B. The biosynthetic gene improved DNA cleavage activity. J Am Chem Soc. 2012;134:13501–9. cluster for the antitumor drug bleomycin from Streptomyces verticil- 4. Chen H, Wang J, Cui J, Wang C, Liang S, Liu H, Wen J. Negative regula- lus ATCC15003 supporting functional interactions between nonri- tion of bleomycins biosynthesis by ArsR/SmtB family repressor BlmR in bosomal peptide synthetases and a polyketide synthase. Chem Biol. Streptomyces verticillus. Appl Microbiol Biotechnol. 2019;103:6629–44. 2000;7:623–42. 5. Radwan HH, Alanazi FK, Taha EI, Dardir HA, Moussa IM, Alsarra IA. 25. Xia M, Huang D, Li S, Wen J, Jia X, Chen Y. Enhanced FK506 produc- Development of a new medium containing date syrup for production tion in Streptomyces tsukubaensis by rational feeding strategies based of bleomycin by Streptomyces mobaraensis ATCC 15003 using response on comparative metabolic profling analysis. Biotechnol Bioeng. surface methodology. Afr J Biotechnol. 2010;9:5450–9. 2013;110:2717–30. 6. Zhu X, Kong J, Yang H, Huang R, Huang Y, Yang D, Shen B, Duan Y. Strain 26. Liu H, Huang D, Wen J. Integrated intracellular metabolic profling and improvement by combined UV mutagenesis and ribosome engineering pathway analysis approaches reveal complex metabolic regulation by and subsequent fermentation optimization for enhanced 6′-deoxy-bleo- Clostridium acetobutylicum. Microb Cell Fact. 2016;15:36. mycin Z production. Appl Microbiol Biotechnol. 2018;102:1651–61. 27. Shirling ET, Gottlieb D. Methods for characterization of Streptomyces spe- 7. McCormick JR, Flärdh K. Signals and regulators that govern Streptomyces cies. Int J Syst Bacteriol. 1966;16:313–40. development. FEMS Microbiol Rev. 2012;36:206–31. 28. Yang D, Teng Q, Dong L-B, Crnovčić I, Huang T, Ge H, Shen B. Genome 8. van der Heul HU, Bilyk BL, McDowall KJ, Seipke RF, van Wezel GP. Regula- mining of Streptomyces mobaraensis DSM40847 as a bleomycin producer tion of antibiotic production in : new perspectives from providing a biotechnology platform to engineer designer bleomycin the post-genomic era. Nat Prod Rep. 2018;35:575–604. analogues. Org Lett. 2017;19:1386–9. 9. van Wezel GP, McDowall KJ. The regulation of the secondary metabolism 29. Garrity GM, Bell JA, Lilburn TG. Taxonomic outline of the prokaryotes. of Streptomyces: new links and experimental advances. Nat Prod Rep. Bergey’s manual of systematic bacteriology. Berlin: Springer; 2004. 2011;28:1311–33. 30. Gröning JA, Tischler D, Kaschabek SR, Schlömann M. Optimization of a 10. Rigali S, Titgemeyer F, Barends S, Mulder S, Thomae AW, Hopwood DA, genome-walking method to suit GC-rich template DNA from biotechno- Van Wezel GP. Feast or famine: the global regulator DasR links nutrient logical relevant Actinobacteria. J Basic Microbiol. 2010;50:499–502. stress to antibiotic production by Streptomyces. EMBO Rep. 2008;9:670–5. 31. Tan G, Gao Y, Shi M, Zhang X, He S, Chen Z, An C. SiteFinding-PCR: a 11. Urem M, Świątek-Połatyńska MA, Rigali S, van Wezel GP. Intertwining simple and efcient PCR method for chromosome walking. Nucleic Acids nutrient-sensory networks and the control of antibiotic production in Res. 2005;33:e122. Streptomyces. Mol Microbiol. 2016;102:183–95. 32. Galm U, Wang L, Wendt-Pienkowski E, Yang R, Liu W, Tao M, Coughlin JM, 12. Craig M, Lambert S, Jourdan S, Tenconi E, Colson S, Maciejewska M, Shen B. In vivo manipulation of the bleomycin biosynthetic gene cluster Ongena M, Martin JF, van Wezel G, Rigali S. Unsuspected control of in Streptomyces verticillus ATCC15003 revealing new insights into its siderophore production by N-acetylglucosamine in streptomycetes. biosynthetic pathway. J Biol Chem. 2008;283:28236–45. Environ Microbiol Rep. 2012;4:512–21. 33. Saito A, Shinya T, Miyamoto K, Yokoyama T, Kaku H, Minami E, Shibuya 13. Nazari B, Kobayashi M, Saito A, Hassaninasab A, Miyashita K, Fujii T. Chitin- N, Tsujibo H, Nagata Y, Ando A. The dasABC gene cluster, adja- induced gene expression in secondary metabolic pathways of Streptomy- cent to dasR, encodes a novel ABC transporter for the uptake of N, ces coelicolor A3 (2) grown in soil. Appl Environ Microbiol. 2013;79:707–13. N′-diacetylchitobiose in Streptomyces coelicolor A3 (2). Appl Environ 14. Rigali S, Nothaft H, Noens EE, Schlicht M, Colson S, Müller M, Joris B, Microbiol. 2007;73:3000–8. Koerten HK, Hopwood DA, Titgemeyer F. The sugar phosphotransferase 34. Inokuma K, Matsuda M, Hasunuma T, Kondo A. Widespread efect of system of Streptomyces coelicolor is regulated by the GntR-family regula- N-acetyl-d-glucosamine assimilation on the metabolisms of amino acids, tor DasR and links N-acetylglucosamine metabolism to the control of purines, and pyrimidines in Schefersomyces stipitis. Microb Cell Fact. development. Mol Microbiol. 2006;61:1237–51. 2018;17:153. 15. Świątek-Połatyńska MA, Bucca G, Laing E, Gubbens J, Titgemeyer F, Smith 35. Pietrocola F, Galluzzi L, Bravo-San Pedro JM, Madeo F, Kroemer G. Acetyl CP, Rigali S, van Wezel GP. Genome-wide analysis of in vivo binding of coenzyme A: a central metabolite and second messenger. Cell Metab. the master regulator DasR in Streptomyces coelicolor identifes novel non- 2015;21:805–21. canonical targets. PLoS ONE. 2015;10:e0122479. 36. Galm U, Wendt-Pienkowski E, Wang L, Huang S-X, Unsin C, Tao M, Cough- 16. Tenconi E, Urem M, Świątek-Połatyńska MA, Titgemeyer F, Muller YA, van lin JM, Shen B. Comparative analysis of the biosynthetic gene clusters and Wezel GP, Rigali S. Multiple allosteric efectors control the afnity of DasR pathways for three structurally related antitumor antibiotics: bleomycin, for its target sites. Biochem Biophys Res Commun. 2015;464:324–9. tallysomycin, and zorbamycin. J Nat Prod. 2011;74:526–36. 17. Nothaft H, Rigali S, Boomsma B, Swiatek M, McDowall KJ, Van 37. Świątek MA, Tenconi E, Rigali S, van Wezel GP. Functional analysis of the Wezel GP, Titgemeyer F. The permease gene nagE2 is the key to N-acetylglucosamine metabolic genes of Streptomyces coelicolor and Chen et al. Microb Cell Fact (2020) 19:32 Page 17 of 17

role in control of development and antibiotic production. J Bacteriol. 44. Okamoto S, Lezhava A, Hosaka T, Okamoto-Hosoya Y, Ochi K. Enhanced 2012;194:1136–44. expression of S-adenosylmethionine synthetase causes overproduc- 38. Liao C, Rigali S, Cassani CL, Marcellin E, Nielsen LK, Ye B-C. Control of chi- tion of actinorhodin in Streptomyces coelicolor A3 (2). J Bacteriol. tin and N-acetylglucosamine utilization in Saccharopolyspora erythraea. 2003;185:601–9. Microbiology. 2014;160:1914–28. 45. Kim W-S, Wang Y, Fang A, Demain AL. Methionine interference in rapa- 39. Resch M, Schiltz E, Titgemeyer F, Muller YA. Insight into the induction mycin production involves repression of demethylrapamycin methyl- mechanism of the GntR/HutC bacterial transcription regulator YvoA. transferase and S-adenosylmethionine synthetase. Antimicrob Agents Nucleic Acids Res. 2010;38:2485–97. Chemother. 2000;44:2908–10. 40. Colson S, van Wezel GP, Craig M, Noens E, Nothaft H, Mommaas AM, 46. Shen B, Du L, Sanchez C, Edwards DJ, Chen M, Murrell JM. Cloning and Titgemeyer F, Joris B, Rigali S. The chitobiose-binding protein, DasA, acts characterization of the bleomycin biosynthetic gene cluster from Strepto- as a link between chitin utilization and morphogenesis in Streptomyces myces verticillus ATCC15003. J Nat Prod. 2002;65:422–31. coelicolor. Microbiology. 2008;154:373–82. 41. Osman D, Cavet JS. Bacterial metal-sensing proteins exemplifed by ArsR– SmtB family repressors. Nat Prod Rep. 2010;27:668–80. Publisher’s Note 42. Rajesh T, Song E, Kim J-N, Lee B-R, Kim E-J, Park S-H, Kim Y-G, Yoo D, Park Springer Nature remains neutral with regard to jurisdictional claims in pub- H-Y, Choi Y-H. Inactivation of phosphomannose isomerase gene abol- lished maps and institutional afliations. ishes sporulation and antibiotic production in Streptomyces coelicolor. Appl Microbiol Biotechnol. 2012;93:1685–93. 43. Yang Y-H, Song E, Park S-H, Kim J-N, Lee K, Kim E, Kim Y-G, Kim B-G. Loss of phosphomannomutase activity enhances actinorhodin production in Streptomyces coelicolor. Appl Microbiol Biotechnol. 2010;86:1485–92.

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